The long term objective of this study is to enhance understanding of the mechanisms regulating tissue factor (TF)-dependent blood coagulation on cell surfaces. A human ovarian carcinoma cell line constitutively expressing cell surface tissue factor will be used for these studies and biochemical reactions will be carried out in purified systems. In addition as indicated other cell lines will also be used as positive and negative controls. Four major questions will be examined. The first aim is to determine whether earlier results that factor VII bound to reconstituted purified TF is preferentially and rapidly activated by trace concentrations of factors Xa and IXa will also hold for factor VII bound to cell surface TF. Additionally, the effect of trace concentrations of other proteases, e.g., factor Xlla and thrombin will be examined. The information obtained from this study may provide clues in understanding why infused factor VIla is effective In controlling the bleeding in hemophiliacs and for an apparent Increased susceptibility to venous thrombosis of patients with factor VII deficiency. The second aim is to examine the importance of asymmetric distribution of negatively charged phospholipids on cell surfaces as a modulator of TF functional expression on cell surfaces. In these experiments various chemical and physiological stimuli will be used to alter the membrane. Annexin V, a protein that binds to negatively charged phospholipids in vesicles and on cell surfaces will be used as a probe to monitor the changes in expression of anionic phospholipid on the cell surface. The third aim is to probe the nature of factor Xa and factor Xa/tissue factor pathway inhibitor (TFPI) complex interaction with cell surfaces. The final aim is to test whether binding of TFPI alone or factor XA/TFPI complex to cell surface factor VIIA/TF leads to internalization of the inhibited complex. In the above proposed studies various biochemical techniques will be employed such as radioligand binding, activation peptide release from tritiated factors IX and X, immunolabelling, SDS-PAGE analysis, and electron microscopy.